Please help, I am new to western blot analysis.

I did a blot using beta-actin antibody and biotinylated protein ladder. I also used an antibody that worked for me before on the other side of the membrane (I cut it in half).

The beta-actin antibody gave no signal, and the ladder was very faint. I did use anti-biotin in my 2nd antibody. I know that my transfer and technique was good because of the other antibody I used, the ladder and protein of interest were correct.

Am I blocking too much? Could degradation of an antibody block signal of the protein ladder?

Thank you.

try
1. increase your protein loading
2. increase your 1st antibody concentration

and,
you can't over-block the membrane

How long and at what temperatures did you leave your primary and secondary antibodies on the blot?

Rb

did you check your transfer with ponceau staining or something similar?
3 years is a little bit for an antibody but it was well stored is not a problen and can be solvented by increasing the amount like R Bishop saids.

1. I doubt the milk would affect things in your case though it can cause problems with phosphorylation antibodies.

2. I would say yes. TRUST NO ONE! Increase the concentration to 1:500 or 1:250. and let us know what you get

Rb

Thank you! I will try your suggestions and get back to you next week.

I am of the mindset of kridos. You may be getting ahead of yourself thinking in terms of antibody problems. Have you first confirmed that you are getting proper transfer? It is especially concerning that you are having problems with your protein ladder, which should be independent of any blocking/staining procedure. Following transfer, check your membrane by staining with Ponceau-S (1% ponceau-S in 10% glacial acetic acid I believe), also you can stain your gel with coomassie. Following the staining, check the gel for the absence of protein (don't be concerned with the appearance of some high molecular weight proteins or uppermost standards) and check your membrane for a nice even transfer. If these are fine, THEN begin to worry about your Western blotting conditions.

what buffer do u use to make the blocking buffer? Does it have any azide in it? If so, then azide with inhibit your secondary antibody and you will see no bands at all. This has happened to me before. Instead you can incubate socondary antibody with TBST for 1 hr at room temperature on a rocker. No milk is requiered. That seemed to work for me.

Try it. I know it can be very frustrating (i was in a similar situation).

Good luck
Pinal